The oncogene c-erbB-2 is known to be associated with the clinical progression of human breast cancer. In vivo models utilizing c-erbB-2's rodent homolog, neu, have been developed to try to evaluate the role of c-erbB-2 in mammary carcinogenesis and tumor biology. In one model transgenic mice have been generated in which the expression of activated neu is targeted to the mammary gland using mammary-specific promoters. In a second model the activated neu oncogene has been directly and stably introduced into in situ rat mammary epithelial cells, using a replication-defective retroviral vector. With both methods, neu was found to be a potent tumor inducer.
We previously have reported "The isolation of a lipocalin uniquely overexpressed in neu-initiated rat mammary carcinomas". S. Stoesz. et al. 1994 AACR Abstract. This lipocalin has been named "NRL" (for neu-related lipocalin). The disclosure of this abstract and of all other publications referred to herein are incorporated by reference as if fully set forth herein. As lipocalins are known to have a wide range of functions, the specific function of NRL is not known.
A protein somewhat homologous to rat NRL, human NGAL, has been isolated and sequenced. Various cDNA gene sequences coding for NGAL and NGAL's protein sequence have been reported in L. Kjeldsen, et al., J. Biol. Chem. 268: 10425-10432 (1993); J. Bundgaard, et al., Biochem Biophys. Res. Comm 202[13]: 1468-1475 (1994); S. Bartsch et al. FEBS Let. 37: 255-289 (1995). NGAL (also known as human neutrophil lipocalin/HNL) has been found in a variety of cell types (e.g. bone marrow; ovarian cell cancers). Again, its specific function is not known.
Note that Bundgaard reported the first base of the mature protein as Q from CAG, whereas Kjeldsen at one location reported an E at that position. The present claims use "NGAL" to cover both variants.
Treatment and diagnosis of breast carcinoma can be improved by a precise determination of the proliferative status of the cancer. One important measure of proliferative status is the percentage of cells in "S-phase". S-phase is the phase of the cell cycle in which duplication of DNA occurs. See generally F. Cross et al., Annu. Rev. Cell Biol. 5:341-395 (1989). Measurement of the percentage of cells in a biopsy sample that are in S-phase is an indicator of cellular proliferation status. A high percentage of cells in S-phase is known to be indicative of a poor prognosis for tumors, absent very aggressive treatment.
The percentage of carcinoma cells in S-phase has been measured by cell staining, flow cytometry, and by analyzing certain markers. Known techniques have problems (e.g. high cost; time consuming) and specific equipment requirements that make the techniques unattractive for routine clinical laboratory usage. Thus, a need exists for an improved assay to determine the proliferative status of carcinomas.